Method of using synthesis of 1,2-dioxetanes and kits therefore

ABSTRACT

Kits comprising 1,2-dioxetanes which can be cause to chemiluminesce by contact with an enzyme, and the enzyme, are provided for use in optically detectable assays. The assay calls for binding the enzyme to the substance to be detected in a sample, removing any unbound enzyme, and then combining the treated sample with the dioxetane. If the substance to be detected is present, enzyme bound thereto will cleave the protecting group of the dioxetane, causing the dioxetane to decompose and chemiluminescence. The intensity of luminescence is indicative of the concentration of the substance in the sample. The substance to be detected may be an enzyme, in which case no binding group is necessary.

This is a continuation of application Ser. No. 08/433,996 filed on May4, 1995 now U.S. Pat. No. 5,679,802.

This application is a continuation-in-part of Edwards, U.S. patentapplication Ser. No. 140,197, filed Dec. 31, 1987.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to assay methods in which a member of aspecific binding pair can be detected and quantified by means of anoptically detectable reaction brought about by the enzymolysis of anenzyme-cleavable group in a 1,2-dioxetane molecule. The inventionrelates specifically to the production of 1,2-dioxetanes and theirintermediate useable in such assay methods.

2. Description of Related Art

1,2-Dioxetanes, cyclic organic peroxides whose central structure is afour-membered ring containing a pair of contiguous carbon atoms and apair of contiguous oxygen atoms (a peroxide linkage), are a known, butheretofore seldom utilized, class of compounds. Because of theirinherent chemical instability, some 1,2-dioxetanes exhibitchemiluminescent decomposition under certain conditions, e.g., by theaction of enzymes, as described in copending, commonly-assignedBronstein, U.S. patent application Ser. No. 889,823 entitled "Method ofDetecting a Substance Using Enzymatically-Induced Decomposition ofDioxetanes", and in copending, commonly assigned Bronstein, et al., U.S.patent application Ser. No. 110,035 entitled "Dioxetanes for Use inAssays", the disclosures of which are incorporated herein by reference.The amount of light emitted during such chemiluminescence is a measureof the concentration of a luminescent substance which, in turn, is ameasure of the concentration of its precursor 1,2-dioxetane. Thus, bymeasuring the intensity and duration of luminescence, the concentrationof the 1,2-dioxetane (and hence the concentration of the substance beingassayed, i.e., the species bound to the 1,2-dioxetane member of thespecific binding pair) can be determined. The appropriate choice ofsubstituents on the 1,2-dioxetane ring allows for the adjustment of thechemical stability of the molecule which, in turn, affords a means ofcontrolling the onset of chemiluminescence, thereby enhancing theusefulness of the chemiluminescent behavior of such compounds forpractical purposes, e.g., in chemiluminescence immunoassays and DNAprobe assays.

The preparation of 1,2-dioxetanes by photooxidation of olefinic doublebonds is known. However, a need exists for a convenient, generalsynthesis of substituted 1,2-dioxetanes from olefinically unsaturatedprecursors derived from readily available or obtainable startingmaterials through tractable intermediates. In this connection, aparticular need exists for a commercially useful method for producingsubstituted 1,2-dioxetanes of the formula: ##STR1## wherein T, R, Y, andZ are defined herein below, from enol ether-type precursors: ##STR2##

Enol ethers can be prepared by several classical methods, for example,by acid-catalyzed elimination of alcohol from acetals R. A. Whol,"Synthesis", p. 38 (1974)!, by Peterson or Wittig reactions ofalkoxymethylene silanes or phosphoranes with aldehydes or ketones inbasic media Magnus, P. et al., Organometallics, 1, 553 (1982)!, and byreactions of alkoxyacetic acid dianions with ketones followed bypropiolactone formation and elimination of CO₂ Caron, G., et al., Can.J. Chem., 51, 981 (1973)!. The O-alkylation of ketone enolate anions isless often used as a general preparative method due to the variableamounts of concomitantly formed alpha-alkylated ketones, the extent ofwhich depends on the solvent, base, alkylating agent and ketonestructure (see, H. O. House, "Modern Synthetic Reactions" pp. 163-215(Benjamin, 1965); and J. D. Roberts and M. C. Caserio, "Basic Principlesof Organic Chemistry" (Benjamin, 1964)). With the use of hexamethylphosphoramide (HMPA), a known carcinogenic solvent, it is, at best,possible to obtain yields of the O-alkylation product which are nohigher than 70%. Moreover, the separation of enol ether from theC-alkylated ketone is quite tedious.

SUMMARY OF THE INVENTION

Adamant-2-yl aryl ketones have been known since the late 1960's (Chem.Abst. 71:P80812V). No attempts to O-alkylate them, however, have beenfound in the literature. It has now been discovered that reaction ofthese ketones, as enolates, with reactive alkylating agents containing"hard" leaving groups see, Fleming, I., "Frontier Orbitals and OrganicChemical Reactions", p. 40 (Wiley, 1976)!, if carried out in a polaraprotic solvent such as dimethyl sulfoxide, dimethylformamide,1,3-dimethyl-2-imidazolidinone, N-methyl-2-pyrrolidone, and the like, ora mixture of such solvents, results exclusively in O-alkylation. Theenol ethers thus obtained can be used as convenient intermediates in thesynthesis of water-soluble or water compatible 1,2-dioxetanes. Suchintermediates can be used to prepare substrates which react with singletoxygen (generated chemically or photochemically) to yield 1,2-dioxetanesof sufficient stability to be useful in subsequent assay techniquesbased on chemiluminescent dioxetane decomposition. This O-alkylationprocess is general and therefore extendable to other cycloalkyl arylketone substrates, which can be synthesized by the reaction of theappropriate secondary cycloalkyl aldehyde with an aryl Grignard reagent,followed by oxidation of the resulting secondary alcohol with Jonesreagent. Preferably, the Grignard reagent is reacted with a secondarycycloalkyl nitrile, followed by acid hydrolysis to form a ketone via animine salt. In all cases, starting materials and products contain afunctional group attached to a secondary carbon atom of the cycloalkylsystem, which is the case of fused polycycloalkyl (e.g., adamantyl)systems if flanked on either side by a bridgehead carbon atom.

It is, thus, an object of this invention to provide novel syntheticroutes to enzyme-cleavable 1,2-dioxetane derivatives.

It is a further object of this invention to provide processes for thepreparation of novel chemical intermediates in the synthesis of1,2-dioxetanes.

Yet another object of this invention is to provide novel compositions ofmatter, such as trisubstituted enolether phosphates, useful as syntheticprecursors of 1,2-dioxetanes which dioxetanes decompose enzymatically inan optically-detectable reaction.

These and other objects of the invention, as well as a fullerunderstanding of the advantages thereof, can be had by reference to thefollowing description and claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Among the 1,2-dioxetanes that can be prepared in accordance with thepresent invention are those having the formula: ##STR3##

In this formula T represents a stabilizing group that prevents thedioxetane compound from decomposing before the bond in the labile ringsubstituent attached to Y is intentionally cleaved, such as an arylgroup, a heteroatom group, or a substituted cycloalkyl group having from6 to 12 carbon atoms, inclusive, and having one or more alkoxy or alkylsubstituents containing from 1 to 7 carbon atoms, inclusive, e.g.,4-tertbutyl-1-methyl-cyclohex-1-yl. The above groups can be used in anycombination to satisfy the valence of the dioxetane ring carbon atom towhich they are attached. Alternatively, T may be a cycloalkylidene groupbonded to the 3-carbon atom of the dioxetane ring through a spirolinkage and having from 5 to 12 carbon atoms, inclusive, which may befurther derivatized with one or more substituents which can be alkyl oraralkyl groups having from 1 to 7 carbon atoms, inclusive, or aheteroatom group which can be an alkoxy group having from 1 to 12 carbonatoms, inclusive, such as methoxy or ethoxy, e.g.,4-tertbutyl-2,2,6,6-tetramethylcyclohexyliden-1-yl. The most preferredstabilizing group is a fused polycycloalkylidene group bonded to the3-carbon atom of the dioxetane ring through a carbon-carbon or a spirolinkage and having two or more fused rings, each having from 3 to 12carbon atoms, inclusive, e.g., an adamant-2-ylidene or an adamant-2-ylgroup, which may additionally contain unsaturated bonds or 1,2 fusedaromatic rings, or a substituted or unsubstituted alkyl group havingfrom 1 to 12 carbon atoms, inclusive, such as tertiary butyl or2-cyanoethyl, or an aryl or substituted aryl group such ascarboxyphenyl, or a halogen group such as chloro, or a heteroatom groupwhich can be a hydroxyl group or a substituted or unsubstituted alkoxyor aryloxy group having from 1 to 12 carbon atoms, inclusive, such as anethoxy, hydroxyethoxy, methoxyethoxy, carboxymethoxy, or polyethyleneoxygroup.

The symbol Y represents a light-emitting fluorophore-forming fluorescentchromophore group capable of absorbing energy to form an excited energystate from which it emits optically detectable energy to return to itsoriginal energy state. Any carbon position in Y can be attached to thedioxetane ring.

Examples of suitable Y chromophores include:

1) phenylene and phenylene derivatives, e.g., hydroxyphenyl,hydroxybiphenyl, hydroxy-9,10-dihydrophenanthrene;

2) naphthalene and naphthalene derivatives, e.g., 5-dimethylaminonaphthalene-1-sulonic acid, hydroxy naphthalene, naphthalimides orhydroxy naphthalimides;

3) anthracene and anthracene derivatives, e.g., 9,10-diphenylanthracene,9-methylanthracene, 9-anthracene carboxaldehyde, hydroxyanthracenes and9-phenylanthracene;

4) rhodamine and rhodamine derivatives, e.g., rhodols, tetraethylrhodamine, tetraethyl rhodamine, diphenyldimethyl rhodamine,diphenyldiethyl rhodamine, and dinaphthyl rhodamine;

5) fluorescein and fluorescein derivatives, e.g., 4- or7-hydroxyfluorescein, 6-iodoacetamido fluorescein, andfluorescein-5-maleimide;

6) eosin and eosin derivatives, e.g., hydroxy eosins,eosin-5-iodoacetamide, and eosin-5-maleimide;

7) coumarin and coumarin derivatives, e.g.,7-dialkylamino-4-methylcoumarin, 4-cyano-7-hydroxy coumarin, and4-bromomethyl-7-hydroxycoumarin;

8) erythrosin and erythrosin derivatives, e.g., hydroxy erythrosins,erythrosin-5-iodoacetamide and erythrosin-5-maleimide;

9) benzheteroazoles and derivatives, e.g., 2-phenylbenzoxazole,hydroxy-2-phenylbenzoxazoles, hydroxy-2-phenylbenzthiazole andhydroxybenzotriazoles;

10) pyrene and pyrene derivatives, e.g., N-(1-pyrene) iodoacetamide,hydroxypyrenes, and 1-pyrenemethyl iodacetate;

11) stilbene and stilbene derivatives, e.g., 6,6'-dibromostilbene andhydroxy stilbenes, hydroxydibenzosuberene;

12) nitrobenzoxadiazoles and nitrobenzoxadiazole derivatives, e.g.,hydroxy nitrobenzoxadiazoles, 4-chloro-7-nitrobenz-2-oxa-1,3-diazol,2-(7-nitrobenz-2-oxa-1,3-diazol-4yl) methylaminoacetaldehyde, and6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoic acid;

13) quinoline and quinoline derivatives, e.g., 6-hydroxyquinoline and6-aminoquinoline;

14) acridine and acridine derivatives, e.g., N-methylacridine,N-phenylacridine, hyydroxyacridines, and N-methylhydroxyacridine;

15) acidoacridine and acidoacridine derivatives, e.g.,9-methylacidoacridine and hydroxy-9-methylacidoacridine;

16) carbazole and carbazole derivatives, e.g., N-methylcarbazole andhydroxy-N-methylcarbazole;

17) fluorescent cyanines, e.g., DCM (a laser dye), hydroxy cyanines,1,6-diphenyl-1,3,5-hexatriene, 1-(4-dimethylaminophenyl)-6-phenylhexaytriene, the corresponding 1,3-butadienes, orany hydroxy derivative of the dienes or trienes;

18) carbocyanine and carbocyanine derivatives, e.g., phenylcarbocyanineand hydroxy carbocyanines;

19) pyridinium salts, e.g., 4(4-dialkylamino styryl) N-methyl pyridiniumsalts and hydroxy-substituted pyridinium salts;

20) oxonols; and

21) resorofins and hydroxy resorofins.

The most suitable Y chromophores are derivatives of benzene ornaphthalene: ##STR4##

The symbol Z represents hydrogen (in which case the dioxetane can bethermally cleaved via rupture of the oxygen-oxygen bond), a chemicallycleavable group such as a hydroxyl group, an alkanoyl or aroyl estergroup, or an alkyl or aryl silyloxy group, or an enzyme-cleavable groupcontaining a bond cleavable by an enzyme to yield an electron-richmoiety bonded to chromophore Y, e.g., a bond which, when cleaved, yieldsan oxygen anion, a sulfur anion, an amine, or a nitrogen anion, andparticularly an amido anion such as a sulfonamido anion.

This moiety initiates the decomposition of the dioxetane into ketone andester fragments. Examples of electron-rich moieties include oxygen,sulfur, amine, etc. The most preferred moiety is an oxygen anion.Examples of suitable enzyme-cleavable groups include enzyme-cleavablealkanoyloxy or aroyloxy groups, e.g., an acetate ester group, or anenzyme-cleavable phosphoryloxy group, oxacarboxylate group,1-phospho-2,3-diacylgyceride group, D-xyloside group, D-fucoside group,1-thio-D-glucoside group, adenosine triphosphate analog group, adenosinediphosphate analog group, adenosine monophosphate analog group,adenosine analog group, α- or β-D-galactoside group, α- or β-D-glucosidegroup, α- or β-D-mannoside group, β-D-fructofuranoside group,β-D-glucosiduronate group, p-toluenesulfonyl-L-arginine ester group orp-toluenesulfonyl-L-arginine amide group.

The symbol R represents a C₁ -C₂₀ unbranched or branched, substituted orunsubstituted, saturated or unsaturated alkyl group, e.g., methyl, allylor isobutyl; a heteroaralkyl or aralkyl (including ethylenicallyunsaturated aralkyl) group, e.g., benzyl or vinylbenzyl; a polynuclear(fused ring) or heteropolynuclear aralykyl group which may be furthersubstituted, e.g., naphthylmethyl or 2-(benzothiazol-2-yl)ethyl; asaturated or unsaturated cycloalkyl group, e.g., cyclohexyl orcyclohexenyl; a N, O, or S heteroatom containing group, e.g.,4-hydroxybutyl, methoxyethyl, or polyalkyleneoxyalkyl; an aryl group; oran enzyme labile group containing a bond cleavable by an enzyme to yieldan electron rich moiety bonded to the dioxetane ring. Preferably, R is amethyl or ethyl group.

One or more of the formula components T, R, Y or Z can also include asubstituent which enhances the water solubility of the 1,2-dioxetanesuch as a carboxylic acid, sulfonic acid or their salts, or a quaternaryamino salt group.

At least one of R and Z, and preferably Z, is an enzyme cleavable group,and preferably an enzyme cleavable phosphate ester or glycosidic acetalgroup.

R may be bonded to Y to form a fused ring fluorophore-forming groupwhich is in turn bonded to the 4-carbon atom of the dioxetane through aspiro linkage and which therefore results in an excited lactone fragmentupon chemical or enzymatic dioxetane decomposition. The required enolethers are obtained by intramolecular O-alkylation of fusedpolycycloalkyl aryl ketone enolates by another substituent, e.g., atoluenesulfonyloxyethyl group, in accordance with the methodologypresented herein.

Y may also be further substituted with one or more electron withdrawinggroups, e.g., perfluoroalkyl having from 1 to 7 carbon atoms such astrifluoromethyl; alkyl or arylsulfonyl such as metylsulfonyl; halogensuch as fluoro or chloro; cyano; nitro; alkoxycarbonyl such as --COOEt;alkanoyl such as --COCH₃ ; amidosulfonyl such as --SO₃ NHAr; or with oneor more electron donating groups such as a branched or unbranched alkylgroup having from 1 to 7 carbon atoms; an alkoxy or aralkoxy grouphaving from 1 to 30 carbon atoms which may contain fused aromatic orfused heteroaromatic rings which are further substituted with heteroatomcontaining moieties, e.g., 2-(5-fluoresceinyl)-ethoxy; an aryloxy grouphaving 1 or 2 rings and which may be further substituted, e.g., phenoxy;a branched or straight chain C₁ -C₇ hydroxyalkyl group, e.g.,hydroxymethyl or hydroxyethyl; an aryl group containing one or morehydroxy substituents or alkoxy substituents having 1 to 7 carbon atoms,e.g., 3,5-diethoxyphenyl; or a heteroaryl group having 1 or 2 rings,e.g., benzoxazole, benzthiazole, benzimidazole or benzotriazole.

Furthermore, by suitably modifying T, R, and Y groups of 1,2-dioxetanes,the stability of the 1,2-dioxetanes and the rate of decomposition of the1,2-dioxetanes can be varied. For example, 1,2-dioxetanes can beattached to various molecules (e.g., proteins or haptens) orimmobilizing supports (e.g., polymer membranes); they can alsoconstitute side chain groups of homopolymers or copolymers.

More particularly, the method for producing 1,2-dioxetanes according tothe present invention comprises the following reaction sequence:##STR5## wherein R¹ can be independently any of the substituents asdefined above for R: W⁻ is an acid anion such as halide (e.g.,chloride); and X and the "R-ylating agent" are as defined below.##STR6##

Step 1, which involves the slow attack of an aromatic Grignard reagenton a nitrile, may be run at reflux in several ethereal solvents such asdiethyl ether (34°), THF (67°), or ethylene glycol dimethyl ether (85°).Thus, although the reaction can be run conveniently over the temperaturerange of 30°-85° C., the use of THF at reflux provides optimumperformance with yields above 90%. As will be understood by one skilledin the art, the use of the analogous organolithium compound to replacethe Grignard reagent is possible in the above scheme, however, it isknown that THF and organolithium compounds (especially n-butyllithium,the metal-halogen exchange reagent) can be incompatible at highertemperatures. Therefore, one can have recourse to the methodologydescribed in Edwards, et al., co-pending and commonly-assigned U.S. Ser.No. 213,672, filed Jun. 30, 1988. The reaction of a fusedpolycycloaldehyde with an aromatic organolitium moiety allows a similarbond construction to be accomplished in diethyl ether over a temperaturerange of -60° to 0° C. This process then provides a convenient lowtemperature counterpart to the nitrile reaction, which requires only afacile, ancillary oxidation to arrive at the same ketonic product.

Step III is best accomplished in the solvents listed using sodium orpotassium hydride or potassium tertbutoxide as the base. This steputilizes reactive alkylating agents to give a kinetic product and can berun conveniently over a temperature range of 0° to 60° C., depending onthe "R"-ylating reagent. Dimethyl or diethyl sulfate are particularlyuseful and inexpensive reagents which display optimum performancebetween 25° and 60° C. In Step IV, the phenolic ether cleavage withsodium thioethoxide may be accomplished with soft nucleophiles such aswith lithium iodide in refluxing pyridine, sodium cyanide in refluxingDMSO, or sodium sulfide in refluxing N-methyl-2-pyrrolidinone areidentical in spirit while having other drawbacks from a commercial pointof view.

Steps V, VI and VII, as indicated herein, may be performed separately orin one operation. The cyclic phosphorichloridate is utilized not onlybecause of its monofunctionality, chemoselectivity, and enolether-compatible deprotection mode, but also because, by virtue ofpseudo-rotation, it is 10⁶ times more reactive than acyclic versions.Thus, in cases where an aromatic hydroxyl group is hindered (e.g., aperi position in a polycyclic, aromatic ring system), or if othersubstituents lower the pKb or nucleophilicity of the enol etheroxyanion, reasonable reaction rates and yields are possible. In benzene,THF, diethyl ether, or DMF, phosphate triester formation with a Lewisbase, or with a preformed alkali metal salt can be effected with all ofthe phosphorochloridates listed over a temperature range of -30° to 50°C.

Subsequently, if a pure monosodium cyanoethyl phosphate ester is desiredthe ring cleavage with alkali cyanide in DMF or DMSO, should be run in anarrow temperature range between 15° and 30° C. In a one pot or in situmode this is not important and the range widens to 60° C. on the highend.

It may be apparent that one can employ phase transfer techniques undercatalysis by quaternary ammonium ions or crown ethers to generate aneven more reactive "naked" cyanide and thus to utilize organic solventsof high volatility (e.g., CH₂ Cl₂), facilitating work-up. Alternatively,the direct use of pure, quaternary ammonium cyanides or sulfinates givesimmediate access to phosphate intermediates or products which containassociated gegenions useful in modifying physical properties such assolubility. Such modifications are within the scope of the processparameters disclosed herein.

Beta-elimination processes brought to bear on the cyanoethyl substitutedphosphate diester may occur under the influence of a wide range ofbases. However, aqueous ammonium hydroxide can be used in vast excessdue to its ease of removal at the end of the process. The cleavage canbe accomplished over a temperature range of 25° to 100° C. At highertemperatures, however, provisions must be made to avoid losses ofgaseous ammonia, and thus, a high-pressure vessel or bomb is required.The preferred temperature range is 35° to 55° C., where the phosphatemonoester product is quite stable, and where simple glassware outfittedwith wired septa can be used as a closed system. Use of alkali metal orquaternary ammonium hydroxides in this step requires close attention tostoichiometry, but as stated above, can provide a variety of mixedgegenion phosphate salts.

While chemical methods of dioxetane formation, e.g., triethylsilylhydrotrioxide, or phosphite ozonide sources of singlet oxygen andtriarylamine radical cation mediated one-electron oxidation in thepresence of triplet oxygen are known, sensitized photooxygenation is aparticularly convenient and forgiving process when reactive olefins areused as substrates. A variety of sensitizing dyes must be used toadvantage, with chlorinated hydrocarbons comprising a preferred class ofsolvents. Reactions are rapid over a temperature range of -78° to 25° C.Low temperatures are not required however for these relatively stabledioxetanes, and in the case of certain phosphate salts, solubility willbe reduced. The ability to manipulate gegenions directly via thesynthetic methodology disclosed or in subsequent ion exchange stepspermits flexibility. The preferred temperature range for allphotoxygenation steps is thus 0° to 10° C.

The foregoing sequences of reactions can be carried out step-by-stepwith isolation of the product of each reaction. However, step VI (alkylcleavage with a nucleophilic acidifying anion such as CN⁻ or organicsulfinate ion) and step VII (deprotection via a beta-eliminationreaction) can be performed advantageously without isolation of theintermediate phosphate ester salt; such intermediate need be isolatedonly when it is desired to confirm its existence.

In steps VI and VII of the foregoing reaction sequence, the cation, M⁺,in the salt used in step VI and the cation, M⁺, in the base used in StepVII can be an alkali metal (e.g., Na⁺), ammonium, or a C₁ -C₇ alkyl,aralkyl, or aromatic quaternary ammonium cation, (NR₄)⁺ (wherein R₄ canbe any or all of an alkyl, e.g., ethyl, aralkyl, e.g., benzyl, or formpart of a heterocyclic ring system, e.g., pyridinium), so that theproducts of steps VII and VIII would be as follows: ##STR7## Inaddition, the quaternary ammonium cation can be connected through one ofits quaternizing groups to a polymeric backbone, as follows: ##STR8## orcan itself be part of a polyquaternary ammonium salt. M⁺ can also be afluorescent onium moiety such as a substituted benzopyrillium or 2-4-dimethylaminostyryl!-N-methylpyridinium counterion.

Within the framework of the foregoing synthesis, the present inventioncomprises a process for producing a compound having the formula:##STR9## wherein T=, R, R¹ and Y are defined above, by reacting acompound having the formula: ##STR10## wherein T is spiro bound at acarbon atom alpha to the carbonyl group, with an alkylating agent (or inmore general terms consistent with the definition of R, an "R-ylatingagent") selected from the group including R-sulfate, toluenesulfonate("Tosylate"), methanesulfonate ("mesylate"), trifluoromethanesulfonate("triflate"), and chloromethyl ethers and trialkyloxonium salts, in abasic, polar, aprotic medium, for example, an alkali metal alkoxide indimethyl sulfoxide.

The invention further provides a process for producing a compound havingthe formula: ##STR11## wherein T, R and Y are as defined above,comprising reacting a compound having the formula: ##STR12## wherein Xis an eletronegative leaving group such as halogen (e.g., chloro), inthe presence of a Lewis base such as a tertiary amine (e.g.,triethylamine) dissolved in an aprotic organic solvent, such as anaromatic liquid (e.g., benzene, toluene), and ether (e.g., glyme,diglyme) or a cyclic ether (e.g., tetrahydrofuran ("THF")).

In a one-pot process, where synthesis of a phosphate triester andsubsequent deprotection to a monoester are done in situ, it isadvantageous to preform an alkali metal salt of the aforementioned Y--OHcompound in a polar, aprotic solvent such as dimethylformamide, usingNaH as the base (see Example 16 below). Addition of thephosphorochloridate affords a solution of the triester which can bedirectly converted (⁻ CN, NH₄ OH) to the monoester in the same reactionmedium.

As an alternative to the use of halophosphate, the analogoushalophosphites, i.e., XPO₂ (CH₂)₂, can be used with subsequent oxidationand irradiation to form the dioxetane directly.

In another aspect, the invention provides a process for producingcompounds having the formulas: ##STR13## wherein T, R and Y are asdefined above, R⁵ can be independent of any of the substituentsdescribed above for R, and R² and R³ are each independently cyano,ortho- or para-nitrophenyl, ortho, para- or ortho, ortho'-dinitrophenyl,comprising reacting a compound having the formula: ##STR14## wherein Xis as defined above, in the presence of a Lewis base such as a tertiaryamine (e.g., a trialkylamine) in an aprotic organic solvent such as anaromatic liquid (e.g., benzene or toluene), an ether (e.g., glyme,diglyme) or a cyclic ether (e.g., THF). As an alternative to the use ofhalophosphates, the analogous nor-oxy compounds (i.e., halophosphites)can be used, followed by oxidation at the phosphorous, deprotection andphotooxidation to the dioxetane. In the case of the cyclic phosphite,dioxetane formation and oxidation at the phosphorous can occursimultaneously in the presence of ³ O₂ /¹ O₂ mixtures found in thephotooxidation reaction.

Preferably, the oxidation described above is effected photochemically bytreating the olefin with singlet oxygen (¹ O₂) in the presence of light.¹ O₂ adds across the double bond to form the dioxetane as follows:##STR15## The reaction is preferably carried out at or below 0° C. in ahalogenated solvent, e.g., methylene chloride. ¹ O₂ can be generatedusing a photosensitizer. As photosensitizers, polymer-bound Rose Bengal(commercially known as Sensitox I and available from HydronLaboratories, New Brunswick, N.J.) and methylene blue (a well-known dyeand pH indicator) or TPP (see Example 17 ) below) can be used.

Within the framework of the foregoing syntheses, the present inventionalso comprises a process for producing a compound of the generalstructure: ##STR16## wherein R, T and Y are as defined above, and Z is aD-sugar molecule linked to Y via a glycosidic linkage, by first reactinga component of the following general structure: ##STR17## wherein Y is aphenyl or naphthyl group, with a tetra-O-acetyl-D-hexopyranosyl halideto produce an intermediate of the following general structure: ##STR18##As will be appreciated by one skilled in the art, there are othermethods available for the synthesis of glycosides as the α or β isomers.The use of the acetoxyhalosugars as glycosyl donors in this particularstereoselective mode is illustrative only.

In the second reaction, the acetate protective groups are removed byhydrolysis to produce the following general structure: ##STR19##

In the third reaction, the photochemical oxidation reaction describedabove is applied to the above intermediate to produce as a product:##STR20## wherein T and X are described above, Y is a fluorophore suchas a phenyl or naphthyl moiety, and Z is a sugar linked to Y via an α orβ glycosidic bond.

The dioxetanes of the invention provide a method for generation of lightin an optically detectable assay method to determine the presence orconcentration of a particular substance in a sample. Examples of suchassays include immunoassays to detect antibodies or antigens (e.g.,hormones such as α or β-hCG, TSH, LH, etc., cancer-associated antigenssuch as AFP and CEA) (enzyme-immunoassay); enzyme assay (e.g., alkalinephosphatases and α- or β-D-galactosidases); chemical assays to detectcations, e.g., potassium or sodium ions; and nucleotide probe assays todetect, e.g., viruses (e.g., HSVI, HTLV III, hepatitis virus,cytomegalovirus), or bacteria (e.g., E. coli).

When the detectable substance is an antibody, antigen, or nucleic acid,the enzyme capable of cleaving group Z of the dioxetane is preferablybonded to a substance (i.e., a substance that binds specifically to thedetectable substance), e.g., an antigen, antibody, or nucleic acidprobe, respectively. Conventional methods, e.g., carbodiimide coupling,are used to bond the enzyme to the specific affinity substance; bondingis preferably through an amide linkage.

In general, assays are performed as follows. A sample suspected ofcontaining a detectable substance (e.g., antigen) is contacted with abuffered solution containing an enzyme bonded to a substance having aspecific affinity for the detectable substance (e.g., antibody). Theresulting solution is contacted with a solid phase, e.g.,antibody-binding beads, to which another substance having the specificaffinity, e.g., antibody, is bound. After incubation for a certainperiod, excess enzyme which is bound to be substance with specificaffinity is then washed away, and a 1,2-dioxetane (substrate) having agroup Z that is cleavable by the enzyme portion is added. The enzymecleaves group Z, causing the dioxetane to decompose into ketone andester moieties; chromophore Y bonded to the ester is thus excited andluminesces. Luminescence is detected using, e.g., a cuvette or cameraluminometer, as an indication of the presence of the detectablesubstance in the sample. Luminescence intensity is measured to determinethe concentration of the substance.

When the detectable substance is an enzyme, a specific affinitysubstance (e.g., antibody) is not necessary. Instead, 1,2-dioxetaneshaving a Z group that is cleavable by the enzyme being detected is used.Therefore, an assay for the enzyme involves adding 1,2-dioxetanes to theenzyme-containing sample, and detecting the resulting luminescence as anindication of the presence of the concentration of the enzyme.

The following examples are intended to illustrate the invention indetail, but they are in no way to be taken as limiting, and the presentinvention is intended to encompass modifications and variations of theseexamples within the framework of their contents.

EXAMPLE 1 3-Methoxyphenyl adamant-2-yl ketone

Magnesium turnings (1.64 g, 0.067 mol) were placed in a flame-driedflask under argon. A small crystal of iodine and 7 ml of drytetrahydrofuran ("THF") (freshly distilled over lithium aluminumhydride) was added. A quantity (7 ml, 0.055 mol) of 3-bromoanisole wasadded by syringe to the slightly agitated suspension of the metal. Anexothermic reaction began after brief heating to 50° C. The flask wasplaced in a water bath at room temperature while THF (33 ml) was addedin a thin stream from an addition funnel. After the exothermic reactionhad subsided, the mixture was refluxed for 45 minutes. A solution of2-cyanoadamantane (8.7 g, 0.054 mol; see, "Organic Syntheses", 57, 8(Wiley, 1977)) or van Leusen, A. M. et al., J. Org. Chem., 42, 3114(1977)) in 50 ml of dry THF was added dropwise over 1.5 hours to therefluxing Grignard reagent. After heating the reaction mixture at refluxtemperature overnight, a yellow suspension was obtained. Ether (50 ml)was added, while the flask and its contents were cooled to an ice bath.Concentrated hydrochloric acid (8 ml, 0.096 mol HCl) was added dropwisewith vigorous stirring over a period of 20 minutes. The precipitate wasseparated by filtration, washed with ether, and dried to obtain 29 g ofthe ketenimine salt as a light, buff-colored, non-hygroscopic powdercontaining some residual magnesium. The salt was suspended in a mixtureof 90 ml of ethanol and 90 ml of concentrated hydrochloric acid andrefluxed for 3 hours, during which time the mixture became considerablythinner. After cooling in an ice bath, the resulting solid was brokenup, separated by filtration, washed to neutrality and dried to obtain13.65 g (93% yield based on 2-cyanoadamantane) of the light gray ketone(m.p. 111°-114° C.). Thin layer chromatography ("TLC") indicated thatthe product was sufficiently pure for subsequent manipulation (R_(f)0.45; Whatman K5F CH₂ Cl₂ : hexanes, 50:50). Recrystallization fromhexanes yielded the captioned compound as prismatic crystals (m.p.113°-115° C.). I.R. (CH₂ Cl₂): 2900 cm⁻¹, 1670 cm⁻¹ (C═O), 1590 cm⁻¹,1575 cm⁻¹. ¹ H-NMR (400 MHz, CDCl₃): δ 1.55-2.05 (m, 12H); 2.30 (s, 2H);3.41 (s, 1H); 3.84 (s, 3H); 7.03-7.40 (m, 4H). These data confirmed thefollowing structure: ##STR21##

EXAMPLE 2 Methoxy(3-methoxyphenyl)methylene adamantane

A quantity (11.3 g, 0.042 mol) of 3-methoxyphenyl adamant-2-yl ketoneobtained according to Example 1 was suspended in 90 ml of molecularsieve-dried (3Å) dimethylsulfoxide (DMSO). Heat was applied to dissolvethe suspended soli. Upon cooling to room temperature with stirring, afine suspension was formed. Potassium tertbutoxide (8.5 g, 0.070 mol)was added under an argon atmosphere. After 5 minutes, a nearlyhomogenous orange solution resulted, which was placed in a water bath at50° C. Dimethyl sulfate (4 ml, 0.042 mol) was added dropwise by syringeover a period of 10 minutes. After 15 minutes of further stirring, anadditional 3.3 ml of dimethyl sulfate (0.034 mol) was added in the samefashion. Subsequently, the colorless solution was stirred overnight atroom temperature. After cooling in an ice bath, 0.5 g of K₂ CO₃ and 125ml of ice water added and the mixture extracted with three 50 mlportions of ethyl acetate. The combined organic fractions were washedwith three portions of water, once with 50 ml of saturated aqueous NaClsolution, and dried over K₂ CO₃. The solvent was removed in vacuo toyield an oil. The oil was dissolved in hexane, and the resultingsolution filtered through Celite and concentrated in vacuo to provide11.5 g (96% yield ) of a viscous, straw-colored oily substance. TLCindicated a clean conversion to an enol ether (R_(f) 0.68; E. Merck Al₂O₃ --CH₂ Cl₂ : hexanes--50:50) with a trace of the ketone startingmaterial. The oil was distilled from K₂ CO₃ (b.p. 148°-150° C., 0.25 mmHg). Under these conditions, slightly yellowing occurred in the stillhead. I.R. analysis of this distillate revealed a small ketoneabsorption band at 1670 cm⁻¹. I.R. (CH₂ Cl_(2l) ): 2900 cm⁻¹, 1670 cm⁻¹(weak), 1600 cm⁻¹, 1590 cm⁻¹, 1580 cm⁻¹, 1570 cm⁻¹, 1095 cm⁻¹, 1080 cm⁻¹; ¹ H-NMR 60 MHz (CDCl₃): δ 1.5-2.0 (m, 12H), δ 2.55 (s, 1H); δ 3.2 (s,1H), δ 3.25 (s, 3H), δ 3.75 (s, 3H), and δ 6.7-7.3 (m, 4H). These dataconfirmed that the structure of the product was: ##STR22##

EXAMPLE 3 Methoxy(3-Hydroxyphenyl)Methylene Adamantane

A solution of methoxy(3-methoxyphenyl)methylene adamantane (14 g, 0.049mol), obtained according to Example 2, in 70 ml of molecular sieve-dried(3 Å) dimethylformamide (DMF) was added under an argon atmosphere to asolution of sodium thioethoxide (7.4 g, 0.88 mol) in the same solvent.The mixture was refluxed for 3 hours. After cooling in an ice bath withstirring, the reaction was quenched with 62 g of NH₄ Cl in 200 ml ofwater. Ethyl acetate (120 ml) and a small amount of ice water wereadded. The aqueous layer was separated and extracted with 75 ml of ethylacetate. The organic extract was washed with four 100 ml portions ofwater, then with saturated NaCl solution (100 ml), and quickly driedover Na₂ SO₄. The solution was filtered and concentrated to an oilysubstance which was then triturated with 50 ml of hexanes. Upon removalof the solvent on a rotary evaporator, a solid separated, which was thentriturated with cold hexanes, filtered and washed with hexanes. Thecrude, off-white phenolic product (13 g) was recrystallized from 5% MeOHin CH₃ CN to yield 10 g of colorless prismatic crystals (m.p. 131°-133°C.). I.R. (CH₂ Cl₂): 3580 cm⁻¹, 3320 cm⁻¹, 2910 cm⁻¹, 1590 cm⁻¹, 1580cm⁻¹, 1440 cm-1. ¹ H-NMR (400 MHz; CDCl₃): δ 1.70-1.91 (m, 12H); 2.58(s, 1H): 3.18 (s, 1H); 3.26 (s, 3H): 5.25 (s, 1H; 6.70-7.20 (m, 4H).These data confirm the following structure: ##STR23##

EXAMPLE 4 Ammonium Sodium 3-(Adamantylidenemethoxymethyl) PhenylPhosphate

A quantity (1.1 g, 0.004 mole) of methoxy(3-hydroxyphenyl)methyleneadamantane, obtained according to Example 3, was dissolved in 15 ml ofmolecular sieve-dried (3 Å) benzene under argon. Triethylamine (0.57 ml,0.004 mole) was added via syringe. The stirred solution was cooled to 0°C. in an ice bath for dropwise addition of2-chloro-2-oxo-1,3,2-dioxaphospholane (0.37 ml, 0.004 mole). After 10minutes in the cold bath, the viscous mixture was slowly warmed to roomtemperature and stirred for 3.5 hours. The benzene was removed in vacuo,and 60 ml of ether was added under argon. The suspension was filteredunder an inert atmosphere, and the resulting solid washed with three 20ml portions of ether. The filtrate was removed in vacuo to yield 1.6 gof the phosphate triester as a colorless, viscous oily substance whichwas moisture sensitive. I.R. (CH₂ Cl₂): 2900 cm⁻¹, 1600 cm⁻¹, 1575 cm⁻¹,1300 cm⁻¹ (P═O). No phenolic OH stretching or C═O (1670 cm⁻¹) absorptionwas present in the I.R. spectrum. TLC showed the absence of the startingmaterial. These data are consistent with the following structure of3-(adamantylidenemethoxymethyl) phenyl ethylene phosphate: ##STR24##

The oily substance obtained above was dissolved in 7 ml of DMF, sodiumcyanide (0.21 g, 0.004 mole) was added, and the mixture stirred for 24hours at room temperature. The solvent of the resulting yellow solutionwas distilled off at 50° C. in vacuo and further removed by chasingseveral times with 2 ml portions of xylene. The residue was trituratedwith ether to produce a gum, which was mixed with CH₂ Cl₂, filtered andstripped in vacuo to yield 1.5 g of a light yellow, amorphous foam. I.R.(CH₂ Cl₂): 2240 cm⁻¹ (weak, CN), 1595 cm⁻¹, 1570 cm³¹ 1, 1475 cm⁻¹, 1275cm⁻¹ (P═O), 1235 cm⁻¹, 1100 cm⁻¹. These data are consistent with thefollowing expected structure of sodium3-(adamantylidenemethoxymethyl)phenyl-2'-cyanoethyl phosphate: ##STR25##

This salt (1.5 g, 0.0035 mole) was dissolved in 5 ml of water.Concentrated ammonium hydroxide (5 ml) was then added dropwise. Thesolution was stirred overnight at room temperature. The resulting whiteslurry was cooled in an ice bath and treated with 30 ml of acetonitrile.Filtration and washing with two 15 ml portions of cold acetonitrileafforded 0.95 g of a hygroscopic, white solid (sintered at 115° C.,melted at 130°-133° C.) after brief drying under vacuum. HPLC (reversephase C18-0.1% ammonium acetate/CH₃ CN) gradient) showed one major peak.I.R. (Nujol): 1595 cm⁻¹, 1575 cm⁻¹, 1245 cm⁻¹, 1200 cm⁻¹, 1095 cm⁻¹,1080 cm⁻¹, 890 cm⁻¹. U.V. (20% MeOH-dioxane) max 260/nm.; ε=10,000. ¹H-NMR (400 MHz, D2O): δ 1.60-1.80 (m, 12H); 2.44 (s, 1H); 2.97 (s, 1H);3.22 (s, 1H; 4.65 (s, HOD); 6.88-7.20 (m, 4H). These data confirmed thatthe structure of the product was: ##STR26##

EXAMPLE 53-(2'-Spiroadamantane)-4-Methoxy-4-(3"-Phosphoryloxy)Phenyl-1,2Dioxetane, Sodium Ammonium Salt

In a large culture tube, 0.065 g (0.00017 mole) of the enoletherphosphate salt, obtained according to Example 4, was dissolved in 25 mlof CHCl₃. A quantity (0.210 g) of methylene blue on silica gel (0.0026 gdye/g SiO₂) was added as a sensitizer. The tube was placed in a silveredDewar flask containing a 250 watt, high-pressure sodium lamp inside awater-cooled immersion well. A piece of 5 ml Kapton® (Dupont) was placedinside the well as a U.V. filter. Ice water was pumped through theapparatus to maintain the sample temperature below 10° C. A continuousstream of dry oxygen was passed into the reaction vessel through acapillary tube. The gas flow was adjusted so as to just maintain auniform suspension of the solid-phase sensitizer. After 25 minutes ofirradiation time, the U.V. (260 nm) absorption of the starting materialdisappeared. The light yellow solution was filtered, evaporated, andreconstituted with 10 ml water. The aqueous sample was filtered througha 0.45 micron nylon filter and chromatographed on a reverse phase, C18preparative HPLC column using a water/acetonitrile gradient. Thefractions showing weak U.V. absorption at 277 nm were combined andlyophilized to provide the dioxetane as a white, cotton-like,hygroscopic solid.

AMPPD Na⁺ NH₄ ⁺ salt did not exhibit a melting point. Instead, sublimingvaporization occurred between 145°-150° C. A solid residue remainedwhich partially decomposes but did not melt below 270° C.

¹ H N.M.R. (D₂ O), ppm): 0.89-1.85 (m, 12H); 2.10 (s, 1H); 2.75 (s, 1H);3.15 (s, 3H); 4.65 (s, HOD-NH₄ ⁺); 7.10-7.36 (m, 4H).

I.R. (Nujol mull, cm⁻¹): 3120, 1970-1790 (weak, broad-NH₄ ⁺), 1640(broad), 1600 (weak), 1580, 1284, 1273, 1122, 980, 895.

The structure of the product was thus confirmed as being: ##STR27##

EXAMPLE 63-(2'-Spiroadamantane)-4-Methoxy-4-(3"-Phosphoryloxy)Phenyl-1,2-Dioxetane,Disodium Salt

Methoxy (3-phosphoryloxyphenyl)methylene adamantane sodium ammonium salt(3.3 g) was dissolved in 15 ml of water containing a drop of pyridine.The solution was slowly run over a 3 cm×25 cm column of Amberlite IR 120(plus) ion exchange resin in the pyridinium salt form (Aldrich ChemicalCo.). Upon elution with distilled water, the fractions showingabsorbance at 260 nm. were combined and lyophilized. A portion of theresulting mono pyridinium slat (1 g, 2.3 mmol) was dissolved in 100 mlof CHCl₃ (dried over Al₂ O₃). The resulting solution was placed in alarge cylindrical tube and treated with 5, 10, 15, 20-tetraphenyl-21H,23H-porphine (2 mg. in 1 ml of CHCl₃). The homogeneous green solutionwas cooled to 0° and pre-saturated with oxygen gas via a sparger tube.The mixture was irradiated under constant O₂ flow in a silvered Dewarflask which also contained a cooled immersion well surrounding a 250watt sodium vapor lamp which was filtered by a single sheet (5 mil) ofDuPont Kapton® polyimide film. The temperature in the Dewar remained at0°-5° C. during a 12 minute irradiation. The solvent was removed invacuo followed by the addition of 100 ml of distilled water containing500 mg of NaHCO₃. The resulting light pink solution was cooled andfiltered through a 0.45 micron Teflon® membrane. The resulting aqueoussolution of dioxetane was subjected to a CH₃ CN--H₂ O gradient on apolystyrene chromatography column, followed by a second pass with a CH₃CN--H₂ O gradient. The resulting solution, which was free of inorganicsalts, was lyophilized to produce 800 mg of a granular, faintly yellow,white solid.

This solid did not exhibit a melting point. Instead, decomposition togive adamantanone as a subliming vapor occurred between 145°-150° C. Asolid residue remained which partially decomposed, but did not meltbelow 270° C.

¹ H N.M.R. (D₂ O): δ 0.85-δ 1.75 (m, 12H includes 2 doublets at 0.85, 1Hand 1.13, 1H); δ 2.15 (s, 1H); δ 2.75 (s, 1H); δ 3.10 (s, 3H); δ 7.10-δ7.35 (m, 4H). ³¹ P N.M.R. (D₂ O; p.p.m. vs H₃ PO₄) δ 1.53, singlet. ¹³C-NMR (400 MHz, D₂ O, p.p.m.): 25.52, 25.68, 31.13, 31.55, 32.13, 32.61,32.98, 34.20, 35.68, 50.31, 98.49 dioxetane), 113.61 (dioxetane), 120.95(broad, low intensity), 121.54, 122.10 (broad, low intensity), 129.37,134.56, 154.29.

When the experiment was repeated (300 MHz in D₂ O at 30° C.), thebroadened lines sharpened and became more intense relative to the lineappearing between them. The sharpened resonances appeared at 120.65 and121.99 p.p.m. This behavior is a clear indication of restricted rotationof the aromatic group. ##STR28## I.R. (Nujol mull): 1600 cm⁻¹ (weak),1580 cm⁻¹, 1285 cm⁻¹, 1275 cm⁻¹, 1120 cm⁻¹ (broad), 980 cm⁻¹, 895 cm⁻¹.

EXAMPLE 7 Methoxy(3-Acetoxyphenyl)Methylene Adamantane

A quantity (1 g, 0.0037 mole) of (3-hydroxyphenyl)methoxymethyleneadamantane, obtained according to Example 3, was suspended in 45 ml ofCH₂ Cl₂ under argon. The mixture was stirred while adding triethylamine(0.6 ml, 0.0043 mole) whereby a colorless solution was formed. Aceticanhydride (0.4 ml, 0.0043 mole) was then added dropwise. The solutionwas stirred at room temperature for 48 hours followed by refluxing for 4hours. The solvent was removed prior to the addition of 40 ml of etherand a small amount of activated carbon. Filtration through Celite andconcentration of the filtrate yielded 1.25 g of an oil which waschromatographed through a small column of silica gel (35 g) using theCH₂ Cl₂ :hexanes (50:50) as eluant. The product (0.800 g) was acolorless oil which was homogeneous on TLC (R_(f) 0.32; Whatman K5F, CH₂Cl₂ :hexanes, 50:50). I.R. (film): 2900 cm⁻¹, 1200 cm⁻¹, 1040 cm⁻¹, 1035cm⁻¹. ¹ H-NMR (acetone-d6): δ 1.9-2.2 (m, 12H), δ 2.45 (s, 3H), δ 2.85(s, 1H), δ 3.45 (overlapping singlets 3H & 1H), δ7.2-7.7 (m, 4H). Thesedata confirmed that the structure of the product was: ##STR29##

EXAMPLE 8 Synthesis of3-(2'-Spiroadamantane)-4-Methoxy-4-(3"-Acetoxy)Phenyl-1,2-Dioxetane

A quantity (0.031 g, 0.0001 mole) of (3-acetoxyphenyl)-methoxymethyleneadamantane, obtained according to Example 7, was dissolved in 19.4 ml ofmolecular sieve-dried acetonitrile. A 10 ml portion of this 5.0 mMsolution and Rose Bengal immobilized on polystyrene beads ("Sensitox")(0.160 g supplied by Polysciences) were placed in a test tube. The tubewas placed at the inside edge of a transparent Dewar flask filled withice water. A flow of dry oxygen was initiated through a capillary whichextended to the bottom of the tube. The sample was then irradiated witha 250 watt, high pressure sodium lamp at a distance of 3 inches (7.62cm) from the outer edge of the flask. The disappearance of the band at260 nm in the UV spectrum was monitored over a 3-hour period. Afterremoval of the sensitizer, the slightly yellow solution was concentratedand chromatographed on a reverse phase, C18 preparative HPLC columnusing 60% acetonitrile/water to 100% acetonitrile gradient. Evaporationof the appropriate fractions provided the dioxetanes as an oil. ¹ HNMR(acetone-d6): δ 1.2-2.1 (m, 12H), δ 2.3(s, 1H), δ 2.4 (s, 3H, δ 3.15 (s,1H), δ 3.35 (s, 3H), δ 7.3-7.8 (m, 4H). These data confirmed that thestructure of the product was: ##STR30##

EXAMPLE 93-(2'-Spiroadamantane)-4-Methoxy-4-(3"-Hydroxy)Phenyl-1,2-Dioxetane

A solution of methoxy(3-hydroxyphenyl)methylene adamantane from Example3 (230 mg, 85 mmol), in 25 ml of dry chloroform, was treated with 0.67mg purified methylene blue dye. The solution was photooxygenated asdescribed for seven minutes. TLC (K5F; 5% ethyl acetate-dichloromethane)revealed that the starting material (Rf 0.46) had been completelyconverted to a weakly absorbing (short wave UV) product (Rf 0.55) whichwas chemiluminescent upon heating the plate to 180° C. The solution wasconcentrated in vacuo and flash chromatographed on a short column ofsilica gel (3.5 cm×10 cm) with 2.5% ethyl acetate in dichloromethane aseluant. The appropriate fractions were rotary evaporated to a slightlyyellow oil. Trituration at 0° C. with 5% ethyl ether in hexanes afforded150 mg of the dioxetane as a yellow tinged, white solid which softenedat 115° C. and melted between 118°-121° C. (rapid temperature ramp).

I.R. (CHCl₃, cm⁻¹): 3590, 3360 (broad), 3000, 2920, 2855, 1597, 1588,1448, 1290, 1175, 1066, 954, 870, 854, 710.

¹ H-NMR (400 MHz, CDCl₃): δ 1.03-1.90 (m, 12H; includes two doublets at1.05, 1H and 1.25, 1H); 2.22 (s, 1H); 3.04 (s, 1H); 3.23 (s, 3H); 5.28(br. s, 1H); 6.98-7.32 (m, 4H).

These data are consistent with the structure as follows: ##STR31##

EXAMPLE 103-(2'-Spiroadamantane)-4-Methoxy-4-(3"-Acetoxy)phenyl-1,2-Dioxetane

(3-acetoxyphenyl) methoxymethylene adamantane (1.15 g., 3.68 mmol.) wasdissolved in 100 ml of CHCl₃ (dried over Al₂ O₃). The solution wasplaced in a large cylindrical tube and treated with 0.3 ml of asaturated solution of purified methylene blue in CHCL₃. The homogeneousblue solution was cooled to 0° and pre-saturated with oxygen gas via asparger tube. The mixture was irradiated under constant O₂ flow in asilvered Dewar flask which also contained a cooled immersion wellsurrounding a 250 watt sodium vapor lamp which was filtered by a singlesheet (5 mil.) of DuPont Kapton polyimide film. The temperature in theDewar remained at 0°-5° during a seven minute irradiation. T.L.C.(Whatman K5F, 50:50 CH₂ Cl₂ - hexanes) showed no starting material, andproduct at R_(f) 0.35. The solvent was removed to yield a blue oil. Upondissolution in 20 ml of 50:50 CH₂ Cl₂ -hexane, some precipitation of adark solid occurred. The suspension was applied to a 8 g. column of finemesh silica gel. Elution under pressure with 100 ml of the same solventyielded 1.3 g. of a light yellow oil upon concentration in vacuo.

¹ H-N.M.R. (CDCl₃): δ 0.98-δ 1.90 (m, 12H); δ 2.15 (s, 1H); δ 2.30 (s,3H); δ 3.04 (s, 1H); δ 3.20 (s, 3H); δ 7.10-δ 7.50 (m, 4H).

A portion of the oily product, when stored at 0°-4° C., slowlysolidified over three weeks. Trituration with petroleum ether (B.P.30°-60° C.) at -20° gave a white solid with a slightly yellow tinge;m.p. 78°-81° C.

I.R. (CHCl₃): 3100 cm⁻¹, 2920 cm-1, 2880 cm⁻¹, 1760 cm⁻¹ (C═O), 1605cm⁻¹ (weak), 1585 cm⁻¹, 1370 cm³¹ 1, 1190 cm⁻¹, 1010 cm⁻¹, 910 cm⁻¹, 900cm.

EXAMPLE 11 6-Methoxynaphth-2-yl Adamant-2'-yl Ketone

Magnesium turnings (1.1 g, 0.045 mol) were placed in a flame-dried flaskunder argon together with a crystal of iodine and 5 ml of dry THF. Thesuspension was heated to 45° C., while a solution of2-bromo-6-methoxynapthalene (7.13 g, 0.03 mol) in 25 ml of dry THF wasadded dropwise. When the exothermic reaction began, the flask wassubmerged in a water bath at room temperature. After the addition wascompleted, the reaction mixture was refluxed for 30 minutes. A solutionof 2-cyanoadamantane (4.85 g, 0.03 mol) was added dropwise over a periodof 30 minutes. The resulting golden brown solution was refluxedovernight, cooled in an ice bath, and diluted with 30 ml of ether.Concentrated HCl (5 ml, 0.06 mol) was then added dropwise with stirring.The resulting precipitate was separated by filtration, washed withether, dried, suspended in a mixture of 35 ml of methoxyethanol and 30ml of concentrated hydrochloric acid, and refluxed for 5 hours. Thesolid was collected by filtration while the suspension was still warm,then washed with water. The crude ketone (7.3 g) was obtained as anoff-white powder. TLC (R₁ 0.39; Whatman K5F, CH₂ Cl₂ :hexanes, 40:60)indicated one major product. Recrystallization from 150 ml of ethylacetate yielded 5 g of buff-colored needles (m.p. 173°-75° C.). I.R.(CH₂ Cl₂): 2900 cm⁻¹, 1665 cm⁻¹ (C═O), 1620 cm⁻¹, 1475 cm⁻¹, 1190 cm⁻¹,1165 cm⁻¹. ¹ H-NMR (400 MHz, CDCl₃) δ 1.56-2.11 (m, 12H); 2.36 (s, 2H);3.58 (s, 1H); 3.94 (s, 3H); 7.10-8.26 (m, 6H). The structure of theproduct was confirmed as: ##STR32##

EXAMPLE 12 (6-Methoxynaphth-2-yl)Methoxymethylene Adamantane

A quantity (3.5 g, 0.011 mol) of 6-methoxynaphth-2-yl adamant-2'-ylketone, obtained according to Example 11, was suspended in 30 ml ofsieve-dried (3 Å) DMSO under argon. Potassium tert-butoxide (2.55 g,0.0202 mol) was added with stirring to give a deep orange solutioncontaining some solid. The flask was placed in a water bath at 48° C.and dimethyl sulfate (1.9 ml, 0.020 mol) added dropwise by syringe overa period of 20 minutes. The decolorized solution was allowed to cool toroom temperature and the resulting suspension was stirred overnight. Themixture was cooled in an ice bath and treated dropwise with 10 ml ofwater. Stirring was continued in the cold for 45 minutes. Theprecipitate was separated by filtration and dried by suction beforebeing washed liberally with water. After drying, a white solid (3.45 g,94%) was obtained having a melting point of 78°-80° C. TLC indicated(R_(f) 0.64; E. Merck Al₂ O₃ hexanes:CH₂ Cl₂, 60:40) one homogeneousproduct along with a trace of starting material. I.R. (CH₂ Cl₂) 2900cm⁻¹, 1620 cm⁻¹, 1600 cm⁻¹, 1480 cm⁻¹, 1030 cm⁻¹, ¹ H-NMR (CDCl₃): δ1.80-2.00 (m, 12H); 2.69 (s, 1H); 3.30 (s, 1H); 3.32 (s, 3H); 3.92 (s,3H); 7.13-7.73 (m, 6H) and confirmed that the structure of the productwas: ##STR33##

EXAMPLE 13 (6-Hydroxynaphth-2-yl)Methoxymethylene Adamantane

A solution of (6-methoxynaphth-2-yl) methoxymethylene adamantane (2.0 g,0.066 mol), obtained according to Example 12, in 20 ml of dry DMF wasadded to a solution of sodium ethanethiolate (1.0 g, 0.012 mol) in 20 mlof the same solvent under an inert atmosphere. The mixture was refluxedfor 2.5 hours. Upon cooling in an ice bath, 150 ml of saturated NH₄ Clwas added to the yellow suspension with vigorous stirring. Ethyl acetate(50 ml) and 20 ml of water are then added. After stirring for 10minutes, the ethyl acetate layer was removed, and the aqueous layerextracted with an additional 50 ml of the same solvent. The combinedorganic extracts were washed with four 20 ml portions of water and oncewith 50 ml of saturated aqueous NaCl solution. The solution was driedquickly over Na₂ SO₄, and concentrated to an orange gum, which was thentriturated several times with hexanes. The gum solidified upon storagein a refrigerator to provide 1.7 g of an off-white solid (m.p. 133°-140°C.). An analytical sample melted at 142°-144° C. after recrystallizationfrom CH₃ CN. TLC (E. Merck Al₂ O₃, CH₂ Cl₂ :hexanes, 50:50) showed theproduct naphthol at the origin, while the starting material (R₁ 0.85)was absent. ¹ H-NMR (400 MHz, CDCl₃): δ 1.79-1.97 (m, 12H); 2.68 (s,1H); 3.30 (s, 1H); 3.32 (s, 3H); 7.08-7.73 (m, 6H); (OH proton exhibitedvariable chemical shift). I.R. (CHCl₃, cm⁻¹): 3580, 3300 (broad), 3000,2900, 2840, 1625, 1600, 1475, 1442, 1385, 1280, 1170, 1078, 1085, 900,880, 810. These data confirm that the structure of the product was:##STR34##

EXAMPLE 14 (6-Acetoxynaphth-2-yl)Methoxymethylene Adamantane

A quantity (0.7 g, 0.00218 mole) of the crude naphthol enol ether,obtained according to Example 13, was stirred with 20 ml of CH₂ Cl₂under argon. Triethylamine (0.35 ml, 0.0025 mole) was added to form alight yellow solution. Acetic anhydride (0.24 ml, 0.0025 mole) was thenadded dropwise. The mixture was refluxed for 24 hours and stripped invacuo to produce an oil which was then dissolved in 30 ml of ether andextracted with water (2×15 ml), saturated aqueous sodium bicarbonatesolution (1×15 ml) and saturated aqueous sodium chloride solution (1×20ml). The organic layer was dried quickly over Na₂ SO₄, followed byrotary evaporation to a light yellow oil which slowly solidified. Thesolid was triturated twice with hexanes at 0° C. to produce 300 mg of awhite solid (m.p. 101°-103° C.). I.R. (CH₂ Cl₂): 2900 cm⁻¹, 1769 cm⁻¹(C═O), 1600 cm⁻¹, 1365 cm⁻¹, 1205 cm⁻¹, 1010 cm⁻¹ and ¹ H-NMR (400 MHz,CDCl₃): δ 1.79-1.97 (m, 12H); 2.34 (s, 3H); 2.66 (s, 1H); 3.30 (s,1H+3H); 7.19-7.83 (m, 6H). These data confirmed that the structure ofthe product was: ##STR35##

EXAMPLE 153-(2'-Spiroadamantane)-4-Methoxy-4-(6"-Acetoxy)naphth-2'-yl-1,2-Dioxetane

Methoxy (6-acetoxynaphth-2-yl)methylene adamantane from Example 14 wasphotooxygenated in the same manner as described in Example 8 above. Theresulting3-(2'-spiroadamantane)-4-methoxy-4-(6"-acetoxy)naphth-2'-yl-1,2-dioxetane,after purification by column chromatography, exhibited the following IRand N.M.R. spectra:

I.R. (CHCl₃, cm⁻¹): 2918, 2856, 1755 (C═O), 1605, 1474, 1453, 1372,1194, 1173, 1070, 925, 913, 897.

¹ H-N.M.R. (CDCl₃, p.p.m.): 0.95-2.0 (m, 12H- includes 2 doublets at0.95, 1H, and 1.18, 1H.); 2.19 (s, 1H); 2.38 (s, 3H); 3.10 (s, 1H); 3.24(s, 3H); 7.30-7.96 (m, 6H).

These data confirm the structure as being: ##STR36##

One hundred μl of a 5×10⁻³ M solution of this dioxetane in acetonitrilewas placed in a cuvette, followed quickly by the addition of 2 ml of 75mM NaOH solution. The slightly cloudy solution was placed in a SpexFluorolog Fluorometer and light emission accumulated over 5 successivescans from 400 to 700 nm. at room temperature. This experiment was thenrepeated exactly using a 5×10⁻³ M acetonitrile solution of thecorresponding 2,7-substituted dioxetane. The chemiluminescent emissionspectra of the two dioxetanes were plotted simultaneously as intensityvs. wavelength. The emission from the 2,7-isomer in this predominatelyaqueous experiment occurred at 555 nm while the less intense emissionfrom the 2,6-isomer occurred at 459 nm in the same medium.

An identical experiment was then performed comparing the emission from3-(2'-spiroadamantane-4-methoxy-4-(7"-acetoxy)naphth-2'-yl-1,2-dioxetaneand 3-(2'-spiroadamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-dioxetane)as 5×10⁻³ M solutions in CH₃ CN. The naphthalene based system emittedlight at 555 nm, while the acetoxyphenyl dioxetane did so at 473 nm.with similar intensity.

EXAMPLE 16 Disodium Methoxy (7-Phosphoryloxy-)Naphth-2yl!MethyleneAdamantane

Sodium hydride (50% in mineral oil, 240 mg, 6 mmol) was added under anargon atmosphere to methoxy (7-hydroxy)naphth-2-yl!methylene adamantane(1.45 g, 4.5 mmol) dissolved in sieve-dried DMF (15 ml). The startingcompound is from Edwards, et al., co pending and commonly-assigned U.S.Ser. No. 213,672, filed Jun. 30, 1988, Example I. The solution wasstirred for 10 min. at room temperature to allow complete sodiumnaphthoxide formation and then cooled to 0° C., at which time 520 μl(5.87 mmol) of 2-chloro-2-oxo-1,3,2-dioxaphospholane (Fluka) were addeddropwise to the suspension. The reaction mixture was slowly warmed toroom temperature over 15 min. to ensure formation of methoxy7-(2-oxo-1,3,2-dioxaphospholan-2-oxy)naphth-2-yl!methylene adamantane.Vacuum-dried sodium cyanide (648 mg, 13.2 mmol) was added as a powder,under argon, followed by stirring at room temperature for 1 hour toeffect in situ ring opening of the cyclic phosphate ester. Uponcompletion of the reaction by TLC analysis (silica gel, 20%EtOAc/hexanes and 30% MeOH/EtOAc) of the reaction products, the solventwas stripped in vacuo while warming gently. The crude monosodium methoxy(7- 2-cyanoethyl!phosphoryloxy)naphth-2-yl!methylene adamantane wasdissolved in 7M NH₄ OH (10 ml) and stirred for 15 hours at 40° C. As thereaction proceeded, the product precipitated as a light yellow gum. Theaqueous solution, still containing desired naphthyl phosphate, was drawnoff and lyophilized to a brown powder after adding 564 mg (6.7 mmol) ofNaHCO₃. The freeze-dried powder and the gummy precipitate were dissolvedtogether in minimal MeOH and then precipitated as flocculent, tancrystals upon addition of acetonitrile. The precipitate was collected ina Buchner funnel, washed with acetonitrile and dried. Evaporation of thefiltrate to a small volume followed by addition of CH₃ CN precipitatedmore naphthyl phosphate, which was collected and washed as describedabove. This procedure was repeated two times to remove all of the crudephosphate from the filtrate. The dried crystal cake was purifed bypreparative HPLC, using an CH₃ CN/H₂ O gradient through a polystyrenecolumn (PLRP-S, Polymer Laboratories). The product fractions werecombined and lyophilized to yield 572 mg (28%) of disodium methoxy(7-phosphoryloxy)naphth-2-yl!methylene adamantane as a white, fluffypowder.

¹ H-NMR (D₂ O, p.p.m.): 1.60-1.83 (12H, m); 2.46 (1H, d, J=0.97 Hz);3.02 (1H, br. s); 3.22 (3H, s); 7.20 (1H, d, J=8.43 Hz): 7.29 (1H, d,J=9.28 Hz); 7.51 (1H, s); 7.65 (1H, s); 7.72 (2H, m).

³¹ P NMR (D₂ O, 85% H₃ PO₄ std., p.p.m.): 0.99 (1P).

EXAMPLE 17 Disodium3-(2'-Spiroadamantane)-4-Methoxy-4-(7"-Phosphoryloxy)naphth-2"-yl-1,2-Dioxetane

A solution of disodium methoxy (7-phosphoryloxy)naphth-2-yl!methyleneadamantane (18.8 mg, 0.042 mmol) and5,10,15,20-tetraphenyl-21H,23H-porphine (TPP, 20 μl of a 2% solution ofCHCl₃ by weight) in 2% MeOH/CHCl₃ (10 ml) was irradiated with a 250 W,high pressure sodium lamp at 10° C. while passing a stream of oxygenthrough the solution. A 5 mil. thick piece of Kapton® polyimide film(DuPont) placed between the lamp and the reaction mixture filtered outunwanted UV radiation. Analytical HPLC (UV detector at 254 nm) showedcompleted dioxetane formation after irradiating 10.5 minutes. Thereaction was also followed by UV spectroscopy with absorption at 255 nmdue to the conjugated vinyl group disappearing upon photoxygenation. Thedioxetane showed one major UV absorption at 230 nm. After evaporatingthe chloroform at 0° C., the residue was dissolved in ice water, passedthrough a 0.46μ filter, and separated by preparative HPLC on apolystyrene column with an acetonitrile/water gradient. The fractionswere frozen and lyophilized at 0° C., yielding 12.1 mg (60%) of thedisodium phosphate dioxetane as a white, fluffy powder.

¹ H-NMR (D₂ O, p.p.m.): 0.69 (1H, d); 0.98 (1H, d); 1.34-1.80 (10H, m);2.11 (1H, d, J=1.35 Hz); 2.77 (1H, d, J=1.96 Hz); 3.08 (3H, s);7.31-7.98 (6H, m).

These data confirm the structure to be as follows: ##STR37##

9.5 μl of a 0.4 mM solution of the above dioxetane in a pH 9 carbonatebuffer (0.05M) was added to 490 μl of the same buffer in a glass tube.This solution was treated with 4×10⁻¹⁴ moles of dialyzed alkalinephosphates (Biozyme; ALPI-11G) in 5 μl of deionized water. The tube wasplaced in a luminometer (Turner 20E) at 29° C. to reveal constant greenlight emission for over 60 minutes.

EXAMPLE 18 3-(Adamantylidenemethoxymethyl)Phenyl β-D-GalactopyranosideTetraacetate

To a solution of methoxy (3-hydroxphenyl)methyleneadamantane (1.21 g,4.48 mmole) from Example 3 in 20 ml of molecular sieve-dried (3 Å)N,N-dimethylformamide was slowly added with stirring 0.188 g (4.7 mmole,Aldrich) of 60% NaH under argon at room temperature. Hydrogen evolutionoccurred immediately as the slightly yellow precipitate of sodiumphenoxide formed. After stirring 30 minutes at room temperature, thesuspension was treated with 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosylbromide (1.987 g, 4.8 mmole), obtained by following the procedure of R.W. Jeanloz and P. J. Stoffyn (Methods Carbohydr. Chem., 1:221-227(1962). The resulting orange, homogeneous solution was stirred for threedays at room temperature and then poured into water (400 ml). Themixture was extracted with 30% EtOAc in hexanes (3×120 ml), dried andconcentrated to give 2.96 g of crude reaction product.

The crude product were separated into two fractions by the filtrationthrough a short silica gel column, eluting with 25-40% EtOAc in hexanes.The less polar mixture (1.05 g) contained mainly the enol ether startingmaterial and a small amount ofmethoxy(3-acetoxyphenyl)methyleneadamantane. The Rf values on TLC(Whatman K5F; 30% EtOAc in hexane) were 0.64 and 0.76 respectively. Thismixture could be treated with NaOMe in MeOH to regenerate the startingmaterial, which was recycled.

The more polar mixture (1.06 g) was composed of the desired arylglycoside (Rf=0.40) and β-elimination product (galactal, Rf=0.36) fromacetobromogalactose and was used for the subsequent deacetylationreaction without further purification.

A spectroscopic sample of aryl β-D-galactopyranoside tetraacetate(purity>85%) was obtained from preparative TLC as a gum. IR (CHCl₃):3020, 2908, 2842, 1748 (acetates), 1596, 1575, 1368, 1230 and 1078 cm⁻¹: ¹ H-NMR (400 MHz, CDCl₃): δ 1.7-2.0 (12H, m, adamantane), 2.01 (3H, s,OAc),2.06 (3H, s, OAc), 2.07 (3H, s, OAc) 2.18 s, OAc), 2.64 (1H, br. s,H-1'b), 3.24 (1H, br.s, H-1'a), 3.28 (3H, s, OMe), 4.07 (1H, t, J=6.7Hz, H-5), 4.21 (2H, d, J=6.7 Hz, H-6), 5.05 (1H, d, J=7.6 Hz, H-1), 5.11(1H, dd, J=10.6, 3.2 Hz, H-3), 5.46 (1H, d, J=3.2 Hz, H-4), 5.50 (1H,dd, J=10.6, 7.6 Hz, H-2), 6.92 (1H, br. d, J=8 Hz, H-4" or H-6"), 6.97(1H, br. s, H-2"), 7.02 (1H, br. d, J=7.8 Hz, H-6" or H-4") and 7.26p.p.m. (1H, dd, J=8.0, 7.8 Hz, H-5"). The 7.6 Hz coupling constant for J(1,2) indicates a glycoside having the β configuration. These dataconfirmed that the structure of the product was: ##STR38##

EXAMPLE 19 3-(Adamantylidenemethoxymethyl) Phenyl-β-D-Galactopyranoside

A solution of the polar mixture (1.06 g), obtained from Example 18 in 10ml of MeOH, was treated with excess NaOMe in MeOH (0.75 ml, Aldrich)under argon. After stirring overnight at room temperature, the reactionwas quenched with solid NH₄ Cl (0.7 g) at room temperature and thenstirred for 30 minutes. After rotary evaporation of the methanol, theresidue was triturated with CHCl₃ and filtered through sand. The solidwas washed with additional CHCl₃ until TLC showed that no additionalU.V. absorbing product was eluted. The combined organic solution wasconcentrated to give a yellow gum which was then filtered through ashort silica gel column, eluting with 5-10% MeOH in chloroform, toafford 0.383 g of yellow gum (Rf=0.25; K5F: 10% MeOH-CHCl₃) with anoverall yield of 17-20% from methoxy(3-hydroxyphenyl)methyleneadamantane. IR (CHCl₃): 3010 (OH), 2996, 2904,2820, 1595, 1574 and 1078 cm⁻¹ ; ¹ H-NMR (400 MHz, CDCl₃): δ 1.65-1.95(12 H, m, adamantane), 2.61 (1H, br. s, H-1'b), 3.18 (1H, br. s, H-1'a),3.23 (3H, s, OMe), 3.62 (1H, m, H-5), 3.81 (3H, m, H-3 and H-6), 4.03(1H, dd, J=9, 8.3 Hz, H-2), 4.19 (1H, br.s, H-4), 4.89 (1H, d, J=7.6 Hz,H-1), 6.88-6.95 (2H, m, H-4" and H-6"), 6.99 (₁ H, s, H-2") and 7.15 ppm(1H, t, J=7.8 Hz, H-5"). These data confirmed that the structure of theproduct was: ##STR39##

An analytical sample, obtained from reverse phase preparative HPLC,exhibited a broad melting point range; sintering at 50°-55° to form acontracted column which became translucent at 89°, and almosttransparent from 96°-99°. The column became transparent at 105°, butstill retained its physical integrity. At 117°-120° the materialcollapse against the glass tube as a viscous, non-mobile melt.

EXAMPLE 203-(2'-(2'-Spiroadamantane)-4-Methoxy-4-(3"-β-D-Galactopyranosyloxyphenyl)-1,2-Dioxetane

In a culture tube, 75.1 mg (0.17 mmole) of the enol ether galactosideobtained from Example 19 was dissolved in 12 ml of 5% MeOH in CHCl₃. Aquantity (0.6 mg) of 5,10,15,20 -tetraphenyl-21H, 23H-porphine (TPP) wasadded as a sensitizer to form a homogenous violet solution. The mixturewas saturated with a stream of dry oxygen through a capillary tube andplaced in a silvered Dewar flask containing a 250-watt high pressuresodium lamp inside a water-cooled immersion well. A piece of 5 mil.Kapton® (DuPont) was placed inside the well as a UV filter. Ice waterwas pumped through the apparatus to maintain the sample temperaturebelow 10° C. The solution was irradiated for 10 minutes under constantO₂ flow, during which time the U.V. absorption at 261 nm (CHCl₃) of thestarting material disappeared and a new peak at 272 nm with a shoulderat 278 nm appeared. The solvent was evaporated at low temperature andthe residue was triturated with 30% CH₃ CN in H₂ O. The aqueous samplewas filtered through a 0.45 micron nylon filter and chromatographed on areverse phase preparative HPLC column using a water-acetonitrilegradient. After lyophilization the dioxetane was collected as a white,cotton-like powder in good yield. In a melting point capillary tube, theproduct began to sinter at 97° and proceeded with significant volumeloss between 102° and 107°. The powder became moist at 110°, finallyproducing a clear sticky gum at 118° C. IR (CHCl₃): 3390 (OH), 3000,2914, 2854, 1582, 1284, 1272, and 1068 cm⁻¹ ; ¹ H-NMR (400 MHz, CDCl₃):δ 0.95-2.07 (13H, m), 2.97 (1H, br.s), 3.11 and 3.13 (3H, two s, OMe),3.65 (1H, br.s), 3.82 (3H, br.s), 4.05 (1H, t, J=7.22 Hz), 4.22 (1H,br.s), 4.89 (1H, d, J=7.3 Hz), and 7.01-7.28 ppm (4H, m). These dataconfirm the following structure for the dioxetane product, which existsas a mixture of two diastereomers at C-4: ##STR40##

What is claimed is:
 1. A kit for the generation of light in a opticallydetectable assay, comprising: (a) a 1,2-dioxetane of the formula:##STR41## wherein n-0-19, wherein Z is a moiety selected from the groupconsisting of a phosphoryloxy, oxacarboxylate,1-phospho-2,3-diacyglyceride, D-xyloside, D-fucoside,1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphateadenosine monophosphate, adenosine, α-D-glucoside, β-D-glucoside,α-D-mannoside, β-D-mannoside, β-D-fructofuranoside, β-D-glucosiduronate,p-toluenesulfonyl-L-arginine ester, and p-toluenesulfonyl-1-arginineamide, andQ is selected from the group consisting of a perfluoroalkylgroup of 1-7 carbon atoms, methylsufonyl, a halogen, cyano, nitro,alkoxy carbamoyl of 1-7 carbon atoms, alkanoyl of 1-7 carbon atoms,branched or straight chain alkyl of 1-7 carbon atom and alkoxy of 1-7carbon atoms, and (b) an enzyme which, when brought into contact withsaid 1,2-dioxetane, cleaves said moiety Z.
 2. The kit of claim 1,further comprising a ligand binder selected from the group consisting ofan antigen, an antibody and a nucleic acid probe.
 3. The kit of claim 2,wherein said enzyme is bound to said ligand binder.
 4. The kit of claim1, wherein said enzyme is selected from the group consisting of alkalinephosphatase and β-galactosidase.
 5. A method of optically detecting thepresence or concentration of a substance in a sample, comprisingcontacting said sample with an enzyme bound to a moiety having aspecific affinity for said substance,removing all said enzyme-specificaffinity complex not bound to any substance in said sample, andcontacting said sample with a 1,2-dioxetane of the formula ##STR42##wherein n=0-19, wherein Z is a moiety selected from the group consistingof a phyosphoryloxy, oxacarboxylate, 1-phospho-2,3-diacyglyceride,D-xyloside, D-fucoside, 1-thio-D-glucoside, adenosine triphosphate,adenosine diphosphate adenosine monophosphate, adenosine, α-D-glucoside,β-D-glucoside, α-D-mannoside, β-D-mannoside, β-D-fructofuranoside,β-D-glucosiduronate, p-toluenesulfonyl-L-arginine ester, andp-toluenesulfonyl-1-arginine amide, and Q is selected from the groupconsisting of a perfluoroalkyl group of 1-7 carbon atoms, methylsufonyl,a halogen, cyano, nitro, alkoxy carbamoyl of 1-7 carbon atoms, alkanoylof 1-7 carbon atoms, branched or straight chain alkyl of 1-7 carbon atomand alkoxy of 1-7 carbon atoms, wherein Z is selected so as to cleavableby said enzyme, and detecting any luminescence generated as a result ofsaid contacting step, wherein luminescence is indicative of the presenceof said substance.
 6. The method of claim 5, wherein the intensity ofsaid luminescence is measured and correlated with the concentration ofsaid substance in said sample.
 7. The method of claim 5, wherein saidspecial affinity moiety is selected from the group consisting of anantigen, antibody and nucleic acid probe.
 8. The method of claim 5,wherein Z is phosphoryloxy and said enzyme is a phosphatase.
 9. Themethod of claim 5, wherein said substance to be detected is selectedfrom the group consisting of a hormone, an antigen, a virus and abacteria.
 10. A method of detecting an enzyme in a sample, comprisingcombining said sample with a 1,2-dioxetane of the formula ##STR43##wherein n=0-19, wherein Z is a moiety selected from the group consistingof a phyosphoryloxy, oxacarboxylate, 1-phospho-2,3-diacyglyceride,D-xyloside, D-fucoside, 1-thio-D-glucoside, adenosine triphosphate,adenosine diphosphate adenosine monophosphate, adenosine, α-D-glucoside,β-D-glucoside, α-D-mannoside, β-D-mannoside, β-D-fructofuranoside,β-D-glucosiduronate, p-tolenesulfonyl-L-arginine ester, andp-toluenesulfonyl-1-arginine amide, andQ is selected from the groupconsisting of a perfluoroalkyl group of 1-7 carbon atoms, methylsufonyl,a halogen, cyano, nitro, alkoxy carbamoyl of 1-7 carbon atoms, alkanoylof 1-7 carbon atoms, branched or straight chain alkyl of 1-7 carbon atomand alkoxy of 1-7 carbon atoms, wherein Z is selected so as to becleaved by said enzyme whose detection is sought, and observing saidsample to detect luminescence, wherein detected luminescence isindicative of the presence of said enzyme.
 11. The method of claim 10,wherein the intensity of said luminescence is measured, and wherein theintensity of said luminescence is indicative of the concentration ofsaid enzyme in said sample.